Children and Snakebite: Snake Venom Effects on Adult and Paediatric Plasma

Snakebite is a globally neglected tropical disease, with coagulation disturbances being the primary pathology of many deadly snake venoms. Age-related differences in human plasma have been abundantly reported, yet the effect that these differences pose regarding snakebite is largely unknown. We tested for differences in coagulotoxic effects (via clotting time) of multiple snake venoms upon healthy human adult (18+) and paediatric (median 3.3 years old) plasma in vivo and compared these effects to the time it takes the plasmas to clot without the addition of venom (the spontaneous clotting time). We tested venoms from 15 medically significant snake species (from 13 genera) from around the world with various mechanisms of coagulotoxic actions, across the three broad categories of procoagulant, pseudo-procoagulant, and anticoagulant, to identify any differences between the two plasmas in their relative pathophysiological vulnerability to snakebite. One procoagulant venom (Daboia russelii, Russell’s Viper) produced significantly greater potency on paediatric plasma compared with adult plasma. In contrast, the two anticoagulant venoms (Pseudechis australis, Mulga Snake; and Bitis cornuta, Many-horned Adder) were significantly more potent on adult plasma. All other procoagulant venoms and all pseudo-procoagulant venoms displayed similar potency across both plasmas. Our preliminary results may inform future studies on the effect of snake venoms upon plasmas from different age demographics and hope to reduce the burden of snakebite upon society.


Introduction
Snakebite is a serious health issue with high public impact, particularly in rural, impoverished regions of the tropics [1][2][3]. In June 2017 the World Health Organization rerecognised snakebite in the highest category (A) of Neglected Tropical Diseases, and global efforts are underway to reduce the burden of snakebite on communities [4][5][6]. Snakebite envenomation can affect any biological target reachable by the bloodstream and thus and 60 healthy female patients aged 19-87 [30]. Age-related differences in the levels of coagulation factors, and subsequent possible alteration on standard coagulation tests, may have serious implications for diagnosis and medical treatment, including for snakebite.
Procoagulant venoms mostly lead to incoagulability (inability to clot due to consumption of fibrinogen) [10] and occasionally cause stroke (cerebral infarcts) in adults [31,32] and children [33]. Children may be more susceptible to stroke due to a likely higher venomto-blood ratio in their small body relative to adults. This situation aligns more closely with the effects observed in small mammalian prey: the highly concentrated procoagulant snake toxins within a small blood volume produce a clot which travels to the brain where it is large enough to occlude a small blood vessel [34][35][36][37].
Most studies that describe differences in snakebite across multiple age demographics rely on retrospective clinical data of patients. Such on-the-ground comparisons are valuable in that the whole-of-body effect of envenomation is considered. However, classic coagulation tests on envenomated patients may be insensitive due to positive feedback loops of the coagulation cascade and the long length of time post-envenomation that these tests are performed [38]. In addition, this type of interrogation cannot exclude confounding factors such as venom dose injected, duration until treatment, management of the snakebite (e.g., extent of physical movement post-bite), and variable health pre-bite. Thus, in vitro work can provide a valuable all-else-equal comparison of the susceptibility of different demographics to snakebite.
There is a need to reduce the burden of snakebite upon society [6] and a need for further study regarding the implications of developmental hemostasis [39]. However, literature directly comparing snake venom effects on paediatric plasma compared to that of adults is, to our knowledge, absent, with studies instead investigating plasma from envenomated patients after the damage has already occurred. Such studies cannot track venom action in real time or control for multiple factors affecting envenomation, such as venom dose or time since envenomation. In this study we report the direct effect of a wide range (15) of medically significant snake venoms upon paediatric plasma, which was pooled from 22 healthy children (ages 1.3-4.9 years old; median 3.3), compared to pooled adult plasma (unknown number of individuals; 292 mL; 18+ years old). Our results provide insights into the relative pathophysiological susceptibility of adult and paediatric plasma to coagulotoxic snake venoms from around the world.

Results and Discussion
We used a coagulation analyser to measure the venom-induced clotting times of adult and paediatric plasma by 15 coagulotoxic snake species from around the world (Table 1). We added snake venom to healthy plasma and observed the immediate and direct effects on two different human plasma types (adult and paediatric) compared to the control. In this way we controlled for the venom concentration, the age of patients, and time since envenomation. While valuable for different purposes, testing plasma from envenomed patients leads to insensitivity to classic coagulation tests [38,40].  The results from three standard coagulation assays on our healthy adult and paediatric plasma batches ( Figure 1) aligned with expectations from the literature (e.g., lower fibrinogen levels in paediatric plasma) [55]. When comparing the average AUC values (Area Under the Curve) for 8-point dilution curves ran in triplicate, which ranged in venom concentration from 0.05 µg/mL to 20 µg/mL, we found that Pseudonaja textilis venom was the most potent venom on both plasmas, with clotting times at the 20 µg/mL venom concentration being 5.3-6.1 s. Remarkably, this approaches Claussian clotting time (3.2 s), a clotting time produced by adding excess thrombin to plasma to rapidly convert all available fibrinogen into fibrin [56]. All venoms affected both plasma types similarly (no significant differences), except for three venoms, one of which is procoagulant in nature (Daboia russelii), and two of which are anticoagulant (Pseudechis australis and Bitis cornuta) ( Table 2). Daboia russelii venom clotted paediatric plasma significantly quicker than adult plasma, indicating a greater drop-for-drop susceptibility of paediatric plasma to this venom. In contrast, P. australis and B. cornuta venoms were significantly less potent on paediatric plasma relative to adult plasma.    Table 2. The nonparametric Kolmogorov-Smirnov test revealed no significant differences between plasma types.
The venom from Daboia russelii was included because this species is a highly medically significant snake that occurs throughout 10 South Asian countries. In combination with its sister species, D. siamensis, these snakes are the leading cause of fatal snakebite in India, Bangladesh, Pakistan, Sri Lanka, Burma and Thailand [57,58]. Despite being sluggish and reclusive, flooding events and habitat invasion by humans bring D. russelii in contact with humans, particularly in plantations, which make up a significant proportion of the human-dominated rural landscape in places such as Karnataka, India [59]. As a result, unfortunately the human-snakebite conflict regarding D. russelii is acute. However, the relative effect of D. russelii venom in children has been largely unknown, with one case report of a 10-year-old girl who developed gross myoglobinuria (occurrence of myoglobin (a protein in muscles) in urine; associated with myotoxicity) [60]. Table 2. Area Under the Curve ± SD values, derived from 8-point dilution curves of venom added to either paediatric or adult human plasma (as per methods in Table 2, and t-test q-values for 1% False Discovery Rate using two-stage step-up method.  Table 2. The nonparametric Kolmogorov-Smirnov test revealed no significant differences between plasma types. The venom from Daboia russelii was included because this species is a highly medically significant snake that occurs throughout 10 South Asian countries. In combination with its sister species, D. siamensis, these snakes are the leading cause of fatal snakebite in India, Bangladesh, Pakistan, Sri Lanka, Burma and Thailand [57,58]. Despite being sluggish and reclusive, flooding events and habitat invasion by humans bring D. russelii in contact with humans, particularly in plantations, which make up a significant proportion of the human-dominated rural landscape in places such as Karnataka, India [59]. As a result, unfortunately the human-snakebite conflict regarding D. russelii is acute. However, the relative effect of D. russelii venom in children has been largely unknown, with one case report of a 10-year-old girl who developed gross myoglobinuria (occurrence of myoglobin (a protein in muscles) in urine; associated with myotoxicity) [60].
We observed a significantly greater potency of D. russelii venom upon paediatric plasma compared with adult plasma in vitro ( Figure 2). This finding suggests that a physiological difference between these two plasmas affects the binding or enzymatic kinetics of the venom toxins. The exact cause of this differential potency of D. russelii venom upon the two plasma types is unclear, but some hypotheses are herein proposed. Daboia russelii venom is known to activate FX (via PIIId SVMPs) and FV (via serine proteases) [61][62][63]. Both of these zymogens have been shown (functionally and immunologically) to occur at lower levels in paediatric plasma compared with adults [28,64]. As such, it may be the case that the venom more quickly activates the fewer zymogen targets available, resulting in the greater observed potency in paediatric plasma. Conversely, lower levels of molecular targets could theoretically result in a reduced venom effect. Another possibility is post-translational modification differences in the zymogens [65] may alter toxin binding. Consequently, future work should (1) add an excess of FX and FV zymogens to paediatric plasmas to observe if this alters D. russelii venom potency and (2)  Another biological aspect of plasma that theoretically may affect the potency of snake venom is the availability of phospholipids in the blood. Phospholipids are negatively charged ions released in vivo by activated platelets that are a required substrate on which some coagulation complexes form during coagulation [50]. Despite platelets being absent in the platelet-poor plasma used in the present study, small amounts (estimated 3%) of phospholipid are still present in plasma [66], and phospholipids were also added into the  Another biological aspect of plasma that theoretically may affect the potency of snake venom is the availability of phospholipids in the blood. Phospholipids are negatively charged ions released in vivo by activated platelets that are a required substrate on which some coagulation complexes form during coagulation [50]. Despite platelets being absent in the platelet-poor plasma used in the present study, small amounts (estimated 3%) of phospholipid are still present in plasma [66], and phospholipids were also added into the assay (Table 2) in the same quantity as standard coagulation assays in hospital settings. Regarding the venoms used in this study and their dependence on endogenous phospholipids for their coagulotoxicity, our previous work showed that D. russelii venom is unusually highly dependent upon phospholipid for the activation of Factor X [49], which is not mirrored by species within the genera Macrovipera, Oxyuranus, or Pseudonaja [49,67]. However, no differences in phospholipid levels have been observed between paediatric and adult plasma [68], suggesting that phospholipid levels do not explain the differential potency of D. russelii venom upon the two plasmas. The observed difference may therefore be related to the mechanism of procoagulant action and requires future work to elucidate.
In contrast to the higher potency of D. russelii venom upon paediatric plasma, we observed a decreased potency of both anticoagulant venoms (P. australis and B. cornuta) on paediatric plasma relative to adult plasma ( Figure 3). The relative potency of all venoms is compared in Figure 4. The mechanism of anticoagulant action by P. australis and B. cornuta appears to be the binding to and inhibition of the prothrombinase complex via PLA 2 s [51,69]. The endogenous activity of PLA 2 s (hydrolysis of phospholipids) was excluded as a possible mechanism for anticoagulation by these venoms because a removal of phospholipids from the prothrombinase-inhibition assay (used in that study and the present study) only resulted in slightly delayed clotting times, compared with complete inhibition of clotting times in the presence of venom [42]. Given that prothrombin (the target of prothrombinase) levels are lower in children than adults [70], there may be less substrate upon which the venom toxins can indirectly affect, thus possibly limiting its potency. However, the zymogen targets of D. russelii venom (FX and FV) also occur at lower levels in paediatric plasma, yet that venom was more potent upon paediatric plasma. Furthermore, no significant differences were observed with several other venoms (D. typus, N. scutatus, O. scutellatus, and P. textilis) that activate prothrombin [71], suggesting that lower zymogen levels in paediatric plasma does not always (or necessarily) affect venom potency.
Another possible explanation for the relatively decreased potency of anticoagulant venoms (P. australis and B. cornuta) on paediatric plasma is the decreased potential in infants and children to convert prothrombin to thrombin [27], perhaps making the venom effects upon prothrombinase less impactful to the system. The prothrombinase complex in children may also have an altered structure such as altered post-translational modifications. Regardless, the mechanism behind the greater potency of D. russelii venom upon paediatric plasma is unknown. Unusually, systemic myotoxicity always occurs in adults but rarely children bitten by Daboia russelii [60]. The reason for this discrepancy between adults and children was also unknown, with the authors offering a possibility relating to body size.
Our paper describes in vitro coagulant activity of venoms upon plasma, which has been shown to correlate with in vivo defibrinogenating effects [72]. However, it is important to note that venom-induced differences have also been shown between paediatric and adult whole blood. For example, platelet aggregation was inhibited by N. scutatus venom more so in paediatric blood than in adult blood [73]. This difference was not observed with Pseudonaja textilis (Eastern Brown Snake) venom. It is important to note that, while blood type has been shown to have minor effects on coagulation clotting times [74], our results do not suggest that ABO blood type of our plasmas (i.e., AB+ for adult; unknown for paediatric plasma but highly likely a mixture due to 21 samples being pooled) impacted the results. If the blood type did affect the observed clotting times, we would expect to observe a similar pattern of difference b/w adult and paediatric plasma across the different snake venoms, yet this was not the case; we observed almost parity between the two plasma types for most venoms, except for a few venoms which is discussed above. lower levels in paediatric plasma, yet that venom was more potent upon paediatric pla Furthermore, no significant differences were observed with several other venoms (D pus, N. scutatus, O. scutellatus, and P. textilis) that activate prothrombin [71], sugge that lower zymogen levels in paediatric plasma does not always (or necessarily) venom potency.  Table 2. Grey lines indicate negative control values without venom. Top slower clotting times (higher y-value) indicate greater coagulotoxic potency in the anticoag assay. Note that one additional dilution point (40 µg/mL) was added to the P. australis cu determine if paediatric plasma would reach maximum clotting time (999 sec) of the machine. B row: quicker clotting times (lower y-value) indicate greater coagulotoxic potency for the ps procoagulant assay. A 1% False Discovery Threshold of unpaired t-tests of AUC values (Ta  Table 2. Grey lines indicate negative control values without venom. Top row: slower clotting times (higher y-value) indicate greater coagulotoxic potency in the anticoagulant assay. Note that one additional dilution point (40 µg/mL) was added to the P. australis curve to determine if paediatric plasma would reach maximum clotting time (999 sec) of the machine. Bottom row: quicker clotting times (lower y-value) indicate greater coagulotoxic potency for the pseudo-procoagulant assay. A 1% False Discovery Threshold of unpaired t-tests of AUC values ( Table 2) revealed q-values shown (bolded = significant). Data points are means of n = 3 replicates ± SE error bars.
While in vitro tests in the lab are informative comparisons in all-else-being-equal scenario, many factors besides the venom toxicity (i.e., drop-for-drop activity levels) can greatly influence the extent and speed of clinical symptoms and therefore the medical outcome of an envenomation [75]. Factors that likely influence clinical envenomations include (but are not limited to): venom dose, fang length, bite site location on the body, snakebite first-aid used (e.g., compression bandage, remaining still), the species envenomated, and body size of the victim. Indeed, given that only three out of 15 venoms affected paediatric plasma significantly differently than adult plasma, these factors probably have a much greater influence on the severity of patient coagulopathy than the venom components and mechanisms alone.   Table 2 (* = significant, i.e., p < 0.05) Another possible explanation for the relatively decreased potency of anticoag venoms (P. australis and B. cornuta) on paediatric plasma is the decreased potential fants and children to convert prothrombin to thrombin [27], perhaps making the ve effects upon prothrombinase less impactful to the system. The prothrombinase com in children may also have an altered structure such as altered post-translational mod tions. Regardless, the mechanism behind the greater potency of D. russelii venom paediatric plasma is unknown. Unusually, systemic myotoxicity always occurs in a but rarely children bitten by Daboia russelii [60]. The reason for this discrepancy bet  Table 2 (* = significant, i.e., p < 0.05).
This point is clearly illustrated in this example: an 80 kg adult is estimated to have around 5.6 L of blood, while a 14.5 kg child (3 years old) has around 1.16 L of blood, or 21% that of adults [76]. As such, once venom entered the bloodstream, it would theoretically be around five times more concentrated in a child than in an adult, merely due to body size. This factor is expected to greatly affect the severity of envenomation, as well as possibly determine the net effect of a procoagulant venom. For example, envenomated prey items such as rats are likely to suffer a stroke from blood vessel occlusion (blockage) [34][35][36][37], but adult humans often succumb instead to venom-induced consumptive coagulopathy (VICC) [38,77,78] due to many micro-clots being formed that are not large enough to cause blood vessel occlusion but still consume blood clotting factors.

Conclusions
The blood circulation system is critical for vertebrate functioning because it maintains haemostasis and distributes nutrients, hormones, electrolytes, immune system cells, and gases throughout the body. When this system is disrupted (e.g., by disease or toxins), devastating effects or even death can occur. Our preliminary study provides new insights into possible differences between adult and paediatric plasma and their effect on snake venom function. While no in vitro test can truly measure the overall functionality of the haemostatic system, our tests provide an all-else-being-equal comparison of the pathophysiological susceptibility of paediatric and adult plasmas to coagulotoxic snake venoms of medical significance. We hope our research informs future studies on the effects of snake venom on paediatric plasma to reduce the burden of snakebite upon society.

Plasma Collection and Handling
Citrated, platelet-poor plasma from two demographics were collected for testing: adult and child. Surplus platelet-poor plasma (292 mL) from an unknown number of healthy adults (18+ years old; AB+; Label #4731976) was provided by the Australian Red Cross, under Research Agreement #18-03QLD-09. The plasma bag (292 mL) was defrosted, pooled, aliquoted, flash-frozen in liquid nitrogen, and stored at −80 • C until required, as per previously established protocols [51]. The same pooling and freezing procedures were carried out for paediatric plasma.
Paediatric platelet-poor plasma was collected from 22 children aged 1.3-4.9 years old (median 3.3; 13 males and 9 females) (unknown blood types) attending the Royal Children's Hospital, Melbourne for minor day surgery who did not have a family history of coagulation disorders (e.g., thrombosis). This is the only paediatric plasma to which we had access. Family history was assessed via a brief interview with the parents of the children. This study was approved by the Royal Children's Hospital Ethics in Human Research Committee (#20031). Informed consent was obtained from the parents of the children. All blood samples were collected and stored in one volume of 0.106 mol L −1 citrate per nine volumes of blood. Samples were then centrifuged at 1450× g for 10 min at 10 • C (Megafuge 1.0R; Heraeus, Thermo Scientific, Karlsruhe, Germany), and platelet-poor plasma was stored at −80 • C until testing.
For testing, one plasma aliquot at a time was thawed for five minutes at 37 • C in a Thermo Haake ARCTIC water bath with a SC150-A40 circulator. Defrosted plasma aliquots were replaced every 30 min at maximum to maintain freshness.
All plasma work was undertaken under University of Queensland Biosafety Approval #IBC134BSBS2015 and University of Queensland Human Ethics Committee Approval #2016000256.

Venom Stocks
All venom and plasma work were undertaken under the UQ approval IBC134BSBS2015. Working stocks of venom were made to 1 mg/mL concentration with 50% glycerol to prevent freezing at −20 • C. Venom protein concentration was determined at 280 nm with a Thermo Fisher Scientific™ NanoDrop 2000 UV-Vis Spectrophotometer (Thermofisher, Sydney, NSW, Australia). To control for individual variation in venoms when comparing across species [79][80][81], for all except one venom (A. bibronii) we were able to make/use pooled samples from 2-8 individuals per venom sample. Samples were pooled either during venom extraction (snakes bit the same vial), or in the lab. To pool individually extracted venom samples ex post facto, we first made the stocks to 1 mg/mL and then transferred equal volumes from each sample into a single, new tube. Venoms included in the study are listed in Table 3. Table 3. Coagulation assays used to compare adult and paediatric plasma *.

Coagulation Tests
Clotting times (seconds) of plasma were automatically measured using a STA-R Max ® analyser (Stago, Asnières sur Seine, France). Measurements occurred via a viscosity-based (mechanical) detection system, whereby opposing magnets oscillate a metal pellet inside the test cuvette (250 µL total volume) until a clot is formed.
To establish baseline clotting time values of our two plasma batches, three standard coagulation assays (Table 2) were performed on both plasma types in the absence of venom. PT is a coagulation screening test which measures, as a whole, activity of the extrinsic and common pathway, including activity of the coagulation factors VII, X, V, II, and fibrinogen. Another test, aPTT, is a general coagulation screening test of the intrinsic pathway, including the coagulation factors XII, XI, IX, VIII, X, V, II, and fibrinogen.
Venom-induced clotting times were used as a measure of susceptibility to the coagulotoxic nature of the venoms included. To test procoagulant and pseudo-procoagulant venoms (the latter makes weak, transient clots [82]), we used an aPTT-based assay ('Procoag. venom', Table 2) to accommodate venom in the assay and remove the clotting catalyst (Kaolin) in the standard aPTT assay so as to not mask the venom action. A separate assay ('Anticoag. venom', Table 2), which uses an FX reagent to attempt to clot the plasma after the venom incubates with the plasma for 120 s, was used to test the potency of anticoagulant venoms on the plasmas. For all dose-response curves, dilution of the venoms with OK buffer was performed automatically by the machine. The venom dilution series was as follows: 1:2, 1:5, 1:12, 1:30, 1:80, 1:160, and 1:400, yielding final cuvette concentrations of venom ranging from 0.05 µg/mL to 20 µg/mL. The most dilute concentration (1:400) was replaced by 1:200 for the thrombin and FXa assays due to dilution constraints of the assay. Specific assay details are listed in Table 2. Venoms were run on either the procoagulant or anticoagulant assay based on our previous work (see Table 3 for all venoms and activities) that determined the venoms were procoagulant or anticoagulant in nature on human plasma.
The experimental workflow went as follows: A quality control (aPTT [83] on adult plasma) was run at the start of each day to ensure the plasma was functioning properly and as per all other experimental days. A new venom solution was made up for each concentration curve replicate, with each dilution-point replicate of the triplicate being run independently of each other. Each venom was run on both plasma types before testing the next venom, ensuring the fairest comparison between plasma types across all venoms.
Reagents were kept at 15-19 • C in the machine during experimentation and otherwise stored at 4 • C. Venom samples were replaced at maximum every 15 min to minimise degradation. Since venoms were made up in 50% glycerol, the blank replacement for venom in the negative control tests was 50% glycerol. All tests were performed in triplicate. The same analyser and reagents were used for all experiments, thereby making the results herein comparable.

Statistical Analyses
All stats tests were performed using GraphPad Prism software (v.7.0). We did not test for normality of data due to only three replicates being present per test. To be conservative, for the standard coagulation assays, Kolmogorov-Smirnov tests were used to test significance using cumulative distributions (rather than ranks).
Area Under the Curve (AUC) values of venom-induced clotting time curves were calculated separately for each curve replicate. The Standard Deviation (SD) values were calculated using the three AUC replicates for each curve. To determine if significant differences between plasma types occurred within each venom, multiple t-test comparisons were made between the three adult AUC values and the three paediatric AUC values for each venom. Although AUC values were derived from a series of 8-pt curves run in triplicate, we still only had three replicates of AUC values. Due to this small sample size of AUC values (N = 3), normality was not tested. Instead, a False Discovery Rate (FDR) approach was used, with a two-stage step-up method of Benjamini and Yekutieli [84]. Since we were interested in major, clear differences between the two plasma types, a desired FDR (Q) of 1% was chosen. Q is the proportion of false discoveries divided by the total number of discoveries. A 1% cut-off is more robust than a 5% value because this value determines the acceptable percentage of discoveries that will prove to be false. In other words, no more than 1% of significant 'discoveries' will be false discoveries (due to random scatter of data) while at least 99% of the discoveries are true differences between the AUC means. Because each row (venom) represented different measures, each row was analysed independently to not assume consistent SD. Although this analysis reduced the degrees of freedom and thus power, it enabled robust analyses with fewer assumptions.